N,N,N'-N'-Tetramethyl-p-Phenylenediamine-Dependent Cytochrome Oxidase Analyses of Bacillus Species

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Abstract
A total of 342 strains belonging to the genus Bacillus were examined for cytochrome oxidase activity by using a quantitative colorimetric N,N,N''N'',-tetramethyl-p-phenylenediamine (TMPD) oxidase assay. The following 3 oxidase reaction groups were identified: (i) a highly oxidase-positive group, whose members oxidized TMPD with specific activities of more than 0.400 .mu.mol/h per mg (dry weight); (ii) an oxidase-negative group, whose members were incapable of oxidizing TMPD (like Escherichia coli); and (iii) an intermediate group of strains designated the oxidase-indeterminate group, whose members oxidized TMPD at rates just slightly higher than the autooxidation rate. Of 293 mesophilic Bacillus strains analyzed 23% were oxidase positive, 47% were oxidase indeterminate, and 30% were oxidase negative. Of 37 thermophilic strains, 68% were highly oxidase positive. All of the psychrophiles tested (13 strains) gave oxidase-indeterminate reactions. Spectral analyses of the membranes of 58 Bacillus strains revealed that there was type c cytochrome(s) in all 39 oxidase-positive and oxidase-indeterminate strains. Cytochrome o was detected in all 58 strains, 27 strains contained cytochromes o and a + a3, and 9 strains contained cytochromes o, a + a3, and d. The remaining 22 strains contained cytochrome o exclusively. Analyses of 19 oxidase-negative strains revealed that 17 lacked type c cytochromes; the exceptions were Bacillus megaterium ATCC 14581T (T = type strain) and Bacillus pantothenticus No. 1T. All 19 oxidase-negative strains contained cytochrome o; 11 of these strains also contained cytochromes o, a + a3, and d, and another 4 strains possessed cytochrome o exclusively as the sole terminal oxidase.